The production of recombinant proteins for therapeutic, nutraceutical or industrial uses has enjoyed great success over the past decade. Different eukaryotic cells and organisms have been shown to be able to produce active protein-based therapeutics. Unfortunately, the high costs frequently derived from low recombinant protein production levels and/or from protein isolation and purification procedures, can invalidate their industrial application. Active research is done to improve both production levels and purification procedures by different approaches.
A new technology based on the fusion of a plant seed storage protein domain with the protein of interest (WO 2004/003207) has been developed to increase the stability and accumulation of recombinant proteins in higher plants. These storage proteins are specific to plant seeds wherein they stably accumulate in protein bodies (Galili et al., 1993, Trends Cell Biol 3:437-442).
The storage proteins are inserted into the lumen of the endoplasmic reticulum (ER) via a signal peptide and are assembled either in the endoplasmic reticulum developing specific organelles called ER-derived protein bodies (ER-PBs) or in protein storage vacuoles (PSV) (Okita and Rogers 1996 Annu. Rev. Plant Physiol Mol. Biol. 47: 327-50; Herman and Larkins 1999 Plant Cell 11:601-613; Sanderfoot and Raikel 1999 Plant Cell 11:629-642). Recombinant storage proteins have also been described to assemble in PB-like organelles in non-plant host systems as Xenopus oocytes and yeast.
Expression of cereal prolamins (the most abundant cereal storage proteins) has been described in Xenopus oocytes after injection of the corresponding mRNAs. This system has been used as a model to study the targeting properties of these storage proteins (Simon et al., 1990, Plant Cell 2:941-950; Altschuler et al., 1993, Plant Cell 5:443-450; Torrent et al., 1994, Planta 192:512-518) and to test the possibility of modifying the 19 kDa α-zein, a maize prolamin, by introducing the essential amino acids lysine and tryptophan into its sequence, without altering its stability (Wallace et al, 1988, Science 240:662-664).
Zeins, the complex group of maize prolamins, have also been produced in yeast with various objectives. Coraggio et al., 1988, Eur J Cell Biol 47:165-172, expressed native and modified α-zeins in yeast to study targeting determinants of this protein. Kim et al., 2002, Plant Cell 14: 655-672, studied the possible α-, β-, γ- and δ-zein interactions that lead to protein body formation. To address this question, they transformed yeast cells with cDNAs encoding these proteins. In addition, those authors constructed zein-GFP fusion proteins to determine the subcellular localization of zein proteins in the yeast cells. The yeast cells, then, were used as a model expression system to study zein properties. It is worth to noting that Kim et al., 2002, Plant Cell 14: 655-672, concluded that yeast is not a good model to study zein interactions because zeins, by themselves, were poorly accumulated in transformed yeast. The yeast cells were also used as a model to study the mechanisms that control the transport and protein body deposition of the wheat storage proteins called gliadins (Rosenberg et al., 1993, Plant Physiol 102:61-69).
Here we show that fusion of a protein sequence that mediates induction of recombinant protein body-like assemblies (RPBLAs), as for instance, prolamins or prolamin domains with a peptide or protein of interest (target) mediates the accumulation of those RPBLAs in cells of organisms such as fungi (which includes yeast), algae and animals. Interestingly, these fusion proteins are stably accumulated in animal cells, inside protein body-like organelles structures.